IgA nephropathy (IgAN), the most common glomerulonephritis worldwide, leads to end-stage renal disease in 20-40% of patients and can reduce life expectancy by up to 10 years, as there is no known cure or disease- specific treatment. Most IgAN patients, regardless of age and ethnicity, have immunologic defects resulting in generation of pathogenic IgA1-containing immune complexes, which ultimately deposit in the kidneys to induce renal injury. These renal immunodeposits likely originate from circulating immune complexes consisting of IgA1 with hinge-region galactose-deficient O-glycans (Gd-IgA1) bound by Gd-IgA1-specific IgG autoantibodies. The long-term goal of this project is to define the underlying mechanisms that lead to the formation of pathogenic immune complexes, so that IgAN-specific treatments can be developed. Our hypothesis is that a molecular- level characterization of Gd-IgA1-specific IgG autoantibodies from IgAN patients coupled with an atomic-level characterization of autoantibodies in immune complexes will significantly advance our understanding of immune-complex formation in IgAN. This information will in-turn provide a basis for development of new disease-specific treatments. Over the past three years, the laboratories of the investigators have utilized biochemical, molecular, structural, and clinical studies to begin characterization of IgAN-specific autoantibodies. We have shown that IgG autoantibodies from patients with IgAN harbor a sequence (amino acids YCSR/K) at the junction of framework 3 and the CDR3 in the variable part of the heavy chain (VH), wherein the serine residue is essential for Gd-IgA1 binding. This serine residue originates from a somatic hypermutation (Ala->Ser) and not from a genetic mutation of a VH germline gene. Our crystallographic studies with an IgAN-derived (YCSK) and a germline-reverted (YCAK) recombinant IgG autoantibody revealed that this seemingly minor difference in the amino-acid sequence had allosteric effects on elements surrounding the serine residue, generating a new surface juxtaposed to the CDR loops. This surface is a potential binding site for part of the Gd-IgA1 hinge-region glycopeptide and is a potential target for the design of IgG autoantibody inhibitors. In this proposal, we will determine the population- and individual-level variability of IgG autoantibodies in IgAN based on VH/VL sequences and Gd-IgA1 binding (Aim 1), determine the structural features of representative IgG autoantibodies and the molecular mechanism of Gd-IgA1 recognition (Aim 2), and develop approaches to block the binding of IgG autoantibodies to Gd-IgA1 (Aim 3). By leveraging our access to biobanked clinical samples, new patients, the new high-throughput approaches for cloning and expression of IgG autoantibodies specific Gd-IgA1, high-resolution methods for structural analyses, and high- throughput testing of inhibitors, our studies have progressed to a stage where molecular-level assessments of the autoantibodies will advance our understanding of the mechanisms that drive IgAN disease. The results of our studies will define disease-specific targets to prevent pathogenic immune-complex formation in IgAN.